CCBE1 is a protein recently discovered to be mutated in patients with a severe inherited form of swelling in their skin and intestines known as "Lymphedema-lymphangiectasia-mental retardation syndrome" or Hennekam Syndrome. In these patients, there is dysfunction of the lymphatics, a network of hollow, blind-end tubes, which collect extravascular fluid from the tissues of the body and return this fluid to the blood circulation. How CCBE1 regulates lymphatic function remains completely unknown. In the zebrafish, a small freshwater fish related to the minnow, mutation of the ccbe1 gene leads to absence of all lymphatics, severe swelling, and ultimately death of the fish. These mutant fish have a striking similarity with zebrafish with disruption of a different gene, vegf-c, which encodes the only factor previously known to specifically stimulate growth of lymphatics. CCBE1, therefore, appears to be a second protein capable of stimulating lymphatic growth, acting side-by-side with VEGF-C. This work seeks to further explore the role of CCBE1 in lymphatic growth using genetically modified mice and cultured cells derived from human lymphatics. These studies will focus on defining the role of CCBE1 in the skin, a tissue severely affected in patients with defects in lymphatic function generally, and in Hennekam Syndrome specifically. In this work, we will first test if CCBE1 alone is sufficient to stimulate lymphatic growth by creating mice that have extra copies of the Ccbe1 gene inserted into their DNA. These additional copies of the gene will be engineered to produce additional CCBE1 protein in the skin only. We will evaluate the lymphatics in the skin of these animals to determine if the additional CCBE1protein is capable of inducing changes in lymphatic in size, number, or function. Secondly, we will we will generate mice with a non-functional Ccbe1 gene. We will use these mice to delete Ccbe1 both in all tissues and then the skin alone, to examine the requirement for CCBE1 in mammalian lymphatic development and in the skin specifically. Finally, we will test the ability of CCBE1 protein to directly promote growth and survival of lymphatic endothelial cells in a culture dish. In this simplified system, we will incubate ccbe1 protein with cells isolated from human skin lymphatic vessels. We will carefully measure changes in cell growth, survival, and internal signaling. PUBLIC HEALTH RELEVANCE: This work will study how CCBE1, a gene mutated in patients with a form of severe swelling of skin and gut, regulates lymphatic growth. We will generate mice with either targeted deletion or increased expression of CCBE1 in the skin. We will also test the effects of the protein on lymphatic endothelial cells in culture.